Akt kinase (Protein Kinase B: hereinafter referred to as Akt) is a serine threonine phosphorylation enzyme that was found in the beginning of the 1990s one after the other by following the homology between virus v-Akt. To date, it has been identified that there are three subtypes of the Akt. These molecules have around 80% homology, and have attracted attention from the beginning in relation to oncogenic transformation. In particular, it has been found and attracted attention that they play a central role to inhibit apoptosis in intracellular cytokine signaling (Genes & Dev., 13:2905-2927, 1999; Annu. Rev. Biochem, 67:481-507, 1998; Biochem. J, 335: 1-13, 1998).
This Akt has an approximate molecular weight of 57 kD, is selectively bound to inositol phosphate in the pleckstrin homology domain (PH domain); and has a function at the N-terminal, which mainly plays a role to define the localization to cell membranes. It also has a phosphorylation kinase domain at the c-terminal. It has been speculated that the binding of PIP3 and the like to PH domain by a signal from Phosphatidylinositol 3-kinase (PI3K) to translocate AKT molecules onto the membrane, and to alter the tertiary structure of Akt are involved in the activation.
Phosphorylations of the both two amino acids, threonine308 (Thr308) and serine473 (Ser473) have been considered to be essential for Akt activation. Wnile it has been known that Thr308 is phosphorylated by phosphoinositide dependent kinase (PDK1), the phosphorylation process of Ser473 has not been fully elucidated; it has been only speculated the possibility that some of uncertain molecules such as integrin linked kinase (ILK) or PDK2 are involved in the phosphorylation process. Further, recently the possibility of autophosphorylation for Ser473 phosphorylation has been reported.
It has known that the activated Akt promotes the phosphorylation of the molecules involved in the apoptosis inhibition. An amino acid sequence in the vicinity of serine/threonine, which is phosphorylated by this Akt, has been known as RXRXXS/T (J. Biol. Chem., 275: 36108-36115). The molecule such as BAD.Caspace 9.FKHRI (forkhead transcription factor) has the above amino acid sequence, and has been known as a substrate of Akt under physiological conditions. Inactive BAD is phosphorylated by Akt; binds to 14-3-3 protein in a phosphorylation-dependent manner; and liberates proteins such as active, Bcl-2 or Bcl-XL, which have apoptosis inhibitory action. It is thought that Akt plays a central role for apoptosis inhibitory control through these known functions and unexplained various targets (Cell, 96:857-868, 1999).
As described above, serine/threonine kinase Akt has a function to phosphorylate serine or threonine residue of intracellular protein specifically, and plays a role to mediate signal transduction to multi-organelle by the phosphorylation function. Further, the phosphorylation function of the Akt regulates a variety of intracellular mechanisms, and involved in the regulation of the various intracellular mechanisms such as, mitosis, cell growth, cell differentiation, control of lipid mentabolisn, immune response, inflammatory response, control of glycogen metabolism. At the same time, this means that the phosphorylation function of the Akt is involved in a wide range of various diseases and disorders such as cancer, obesity, autoimmune injury, inflammation and diabetes (type 2).
Recently, it has been reported that Akt activation is involved in breast cancer, lung cancer, prostate cancer, ovarian cancer, or hematological malignancies such as leukemia and lymphoid tumor (Annu. Rev. Biochem. 68, 965, 1999). As Akt activity is raised in these malignancies, Akt activation is considered to be a cause of these malignancies. In recent days, these serine/threonine kinase activities are modulated by using short peptides that are derivatives of the HJ loop of a serine/threonine kinase, with which treatments for diseases and disorders as described above have been attempted (published Japanese translation of PCT international publication No: 2002-500649).
On the one hand, TCL1 has been known as a protooncogene. TCL1 was noted that the activity is raised in human T cell prolymphocytic leukemia (T-PLL), and has been known to date that there are three similar subtypes (TCL1, MTCP1, TCL1b) (Oncogene, 8:2475-2483, 1993; Proc. Natl. Acad. Sci. USA, 91:12530-12534, 1994). It has been known that the expression was activated by translocation of these loci: 14q, 32, χ28 to T cell receptor loci to develop human leukemia (T-PLL). However, it is a small protein of 13-16kD, and does not have the specific function structure that has been known to date, whose function has so far not been elucidated.
The expression of these molecules is comparatively limited under physiological conditions. TLC1 expression is limited to the lymphoid cells of T cells in an early differentiation (CD3-/CD4-/CD8-), and various B cells before the plasma cell differentiation. Further, although the details in the expression of MTCP1 under physiological conditions are unknown, it was identified from the recent analysis results of gene expression that the expression are induced in activated T cells. TCL1b is a recently cloned molecule and exists in the immediate vicinity of TCL locus. It is thought that there are five subtypes in mouse and is only one subtype in human. It has been reported that this gene expression has distinctly high expression in germinal cells in an early differentiation.
Genes of TCL1 are cloned, and a base sequence of 342 and an amino acid sequence of 113 are elucidated (U.S. Pat. No. 5,985,598).
However, functions of TCL1 have been unknown at all to date. The present inventors found that protooncogene TCL1 binds to Akt, from the search result of the protein molecules binding to Akt with the use of a library derived from human B cell by two-hybrid screen using a yeast to elucidate the process of Akt activation. That is, the present inventors indicated that TCL1 is bound to Akt, multimerized, and Akt of the multimer is activated; and found that TCL1 is the Akt co-activator promoting the Akt activation (Mol. Cell, 6:395-407, 2000). Further, the present inventors elucidated that TCL1 facilitated the Akt-mediated cell division, inhibition of apoptosis and the like, and is a predisposing factor for leukemia, tumor of human lymphatic system and the like. In subsequent studies, by coimmunoprecipitation assay using recombinant proteins in and out of cells, the present inventors indicated that TCL1 facilitated the polymerization among heterogeneous Akt molecules and phosphorylation of serine472/473 residues of Akt among heterogeneous Akt molecules; and elucidated the molecular mechanism that TCL1 activates the Akt (J. Biological Chemistry, 277[5], 3743-3751, 2002).
Further, the present inventors prepared an amino acid random library of TCL1 oncogene by applying PCR method and identified the amino acid sites required for the binding of Akt to TCL1 and the polymerization of TCL1, and moreover identified the mutated form of TCL1 which lacks dimerization or binding potency to Akt of TCL1. In addition, it was identified that the mutated form of TCL1 lacks Akt activation potency (both in vitro and in vivo), and loses various types of functions in TCL1 such as stabilization of mitochondrial outer membrane, inhibition of apoptosis, and nuclear translocation of Akt (Molecular and Cellular Biology, 22[5], 1513-1525, 2002). In other words, the present inventors found that protooncogene TCL1, whose function was unknown so far, is a co-activator of Akt, and is essential for the binding to Akt and the polymerization of TCL1-to-TCL1 when Akt activates.
Patent document 1: Published Japanese translation of PCT international publication No. 2002-500649
Patent document 2: Specification of U.S. Pat. No. 5,985,598
Nonpatent document 1: Genes & Dev., 13: 2905-2927, 1999
Nonpatent document 1: Annu. Rev. Biochem, 67: 481-507, 1998
Nonpatent document 1: Biochem. J, 335: 1-13, 1998
Nonpatent document 1: J. Biol. Chem., 275: 36108-36115
Nonpatent document 1: Cell, 96: 857-868, 1999
Nonpatent document 1: Oncogene, 8:2475-2483, 1993
Nonpatent document 1: Proc. Natl. Acad. Sci. USA, 91: 12530-12534, 1994
Nonpatent document 1: Mol. Cell, 6:395-407, 2000
Nonpatent document 1: J. Biological Chemistry, 277[5], 3743-3751, 2002
Nonpatent document 1: Molecular and Cellular Biology, 22[5], 1513-1525, 2002